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Oligo- sulphated disaccharides inhibit human T-lymphocyte tumour necrosis factor (TNFα) synthesis and monocyte differentiation into TNFα synthesis competent macrophages. Low molecular weight heparinoids inhibit various inflammatory responses (Lever & Page, 2012), mainly through inhibition of neutrophil function. Sulphated heparin disaccharides (HDS) are released during inflammation (Cahalon et al., 1997) and inhibit adjuvant arthritis, primary macrophage (Mφ,) and primary T-cell TNF synthesis. We have found that sucrose octasulphate (SOS) and the novel sulphated DS N-acetyl-diglucopyranosyl-amine oligosulphate (diGAS) inhibit rat collagen and murine antigen induced arthritis (Jones et al, 2008), and variably inhibit whole blood but not lps-induced PMA-U937 Mφ TNFα synthesis. We investigate sulphated disaccharides (SDSs) on T-cell and monocyte differentiation to TNFα synthesis-competent Mφ. Jurkat or U937 in antibiotic free, FCS and glutamine supplemented RPMI-1640 were seeded into 96 well plates (105/well). Jurkat cell TNFα synthesis was induced by PHA (1μg/mL, 18hrs). U937 cells were differentiated to Mφ with phorbol-myristate acetate (PMA, 80nM, 72hrs, Yousaf et al, 2012). TNFα synthesis was stimulated with lps (1ug/mL, 5hrs). Drugs: SOS (TC, Canada), DSIII-H & HDSII (Sigma), diGAS (synthesised by Synovo GmbH). Cell viability assessed by trypan blue exclusion. PHA induced TNFα synthesis in Jurkat cells was inhibited by SOS, HDS and diGAS (Table 1). Incubation of U937 cells with the drugs for 2hr prior to the addition of PMA reduced TNFα synthesis (Table 1). However, administered 2hr after PMA resulted in no TNFα synthesis inhibition. Visualisation of cells revealed a lower proportion of adherent cells.
Table 1. Conditions for the inhibition of T-lymphocyte (n=3) and M φ TNFα (n=4) synthesis by SDSs. Dunnett’ s test*=p<0.05, **=p<0.01; ***=p<0.001 TNFα synthesis inhibition by SDS can be replicated in cell lines. These properties are dependent on the time of administration w.r.t. stimulus, and possessing bell-shaped concentration response curves (Cahalon et al 1997). In addition, the non-heparin SOS, and novel DiGaS oligosulphated SDS are effective, compared to the site specific sulphation of HDS. A further finding is the inhibition of monocyte differentiation into Mφ, leading to a new avenue for SDS mechanistic explanation for their anti-rheumatic activities. Further research will determine patterns of Mφ cell marker expression such as CD19. Cahalon L et al, Int Immunol 9:1517, 1997 Jones MR et al, Inflammation Res 57(S2):S105, 2008. Lever R & Page C Handb Exp Pharmacol 207: 281-305. Yousaf N et al, Inflammation Res 61(S1):S24
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